Method for transferring molecules in vital cells by means of laser beams and arrangement for carrying out said method

ABSTRACT

The invention relates to an optical method for targeted transfer of molecules, preferably of DNA, RNA, peptides, amino acids and proteins, into vital cells by means of laser radiation and to an arrangement for implementing the method. The object of the invention, to find a novel possibility for targeted molecule transfer into the interior of vital cells, particularly the transfer of DNA, RNA, peptides, amino acids and proteins, which achieves a high transfer efficiency while extensively excluding destructive side effects such as a lethal effect on a treated cell, is met according to the invention in that cellular membranes are opened transiently for the molecule transfer by multiple laser pulses in the microjoule range or less and a pulsed, near-infrared laser beam with a pulse width in the femtosecond range is directed in each instance to a submicrometer spot of a membrane of the vital cell for an irradiation period of less than one second.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority of PCT Application Serial No.PCT/DE03/01708, filed May 22, 2003, German Application No. 102 23 922.3,filed May 23, 2002 and German Application No. 102 23 921.5, filed May23, 2002, the complete disclosure of which is hereby incorporated byreference.

BACKGROUND OF THE INVENTION

a) Field of the Invention

The invention is directed to an optical method for the targeted transferof molecules, preferably DNA, RNA, peptides, amino acids and proteins,into vital cells by means of laser radiation and to an arrangement forimplementing the method.

The method is advantageously suitable for the transfection of plantcells, animal cells and human cells, for example, for producing drugssuch as synthetic vaccines.

By means of the arrangement according to the invention, the method canbe used effectively for the transfection of genes in individual cellsand opens up applications in the fields of plant materials and animalmaterials production, gene therapy and in the production and applicationof specific drugs, particularly synthetic vaccines.

b) Description of the Related Art

Targeted molecule transfer plays an important role in the production ofvaccines, among other things. Vaccines are used within the framework ofactive immunization of humans and animals for stimulating the immunesystem against pathogenic microorganisms and pathogenic substances.Normally, inactivated or attenuated germs which still retain animmunogenic effect are used. A slight health risk is involved inactivating individual attenuated germs. This may have fatal consequencesparticularly for patients with weakened immune defense. Considerablymore critical, however, is the fact that there is currently no vaccineavailable for many diseases, including AIDS. Methods in gene technologyaim at making possible the production of effective, highly-puresynthetic vaccines through DNA transfer. The implantation of foreign DNAin plants and animals for expressing vaccines in foodstuffs is beingdiscussed. In principle, a specific immunity against very particularamino acid sequences can be achieved.

The transporting of foreign DNA to a target cell can be carried out bymeans of specific carriers, e.g., colloidal particles such asnanospheres and microspheres, emulsions and liposomes. Virus-likeaggregates (VLA) in which the molecules to be transported are enclosedby a two-layer membrane are currently being researched.

When colloidal particles of this kind are applied intravenously, forexample, they are normally intercepted with high efficiency by thereticuloendothelial system (Kupffer cells, etc.) and, therefore, cannoteffect the transfer to the actual target. In order to prevent thisunspecific binding, the surface of the particles is changed, e.g.,through determined coatings and suitable particle sizes, by means ofcomplicated particle engineering. Finally, when the target cell in thetarget organ is reached more or less effectively and selectively,problems arise with respect to particle reception and there is a risk ofdestruction through lysosomal enzymes and nucleases.

Therefore, an efficient foreign DNA accumulation in the cytoplasm ordirectly in the cell nucleus of a specific target cell is desirable.

The targeted transfer of molecules, preferably of DNA into vital cells,was carried out heretofore:

(i) by mechanical processes such as microinjection and particle gunbombardment;

(ii) by means of biological (viruses, bacteria, etc.) and synthesizedcarrier molecules; or

(iii) by permeabilizing the membrane by means of electrical fields(electroporation) or chemical agents (e.g., streptolysin O toxin).

There are problems with all three of these methods with respect to theefficiency of the molecule transfer and the high probability of anunintended lethal effect as is confirmed by the prior art mentioned inthe following.

It is known (e.g., D. J. Stephens, R. Pepperkok: The many ways to crossthe plasma membrane, Proc. Nat. Acad. Sci. USA, 89 (2001) 4295-4298; D.Luo, W. M. Saltzman: Synthetic DNA delivery systems, Nature Biotechnol.18 (2000) 33-37; L. Bildirici, P. Smith, C. Tzavelas, E. Horefti, D.Rickwood: Transfection of cells by immunoporation, Nature 405 (2000)298) to carry out a transfer of molecules, preferably of DNA, into vitalcells by means of synthesized carrier molecules and biological carriersystems (viruses, etc.).

It is likewise known to enable the targeted transfer of moleculesthrough mechanical methods such as microinjection and particle gunbombardment (e.g., M. Knoblauch et al.: A galinstan expansionfemtosyringe for microinjection of eukaryotic organelles andprokaryotes, Nature Biotechnol. 17 (1999) 906-909).

Further, the transfer of molecules into vital cells by permeabilizationof the membrane by means of electrical fields (electroporation) orchemical agents (e.g., streptolysin O toxin) is known.

In conventional vaccine production using gene technology, cell culturesare typically infected with viruses, as carriers of the DNA in question,under strict safety measures in bioreactors; the viruses are theninactivated or attenuated.

The direct transfer of individual molecules in a specifically selectedindividual cell is only possible by means of mechanical microinjectionusing thin glass cannulas (typical distal diameter: 0.5 mm) throughinvasive disruption of the cell membrane; this process is inefficientand entails a high potential of injury. Mechanical gene transfer bymanual microinjection requires specially trained personnel and is met byconsiderable difficulties when transferring into nonadherent cells, inplant cells because of the sturdy cell wall, and with isolatedprotoplasts. In addition, the presence of the glass in the interior ofthe cell causes considerable problems due to the adherence ofintracellular molecules, e.g., certain proteins. The occurringmechanical forces result in additional destructive effects.

Optical methods for targeted molecule transfer based on focused laserradiation through the microablation of a membrane section were carriedout heretofore by means of ultraviolet laser sources with pulse widthsin the nanosecond range and high energy in the microjoule and millijoulerange. Laser pulses with such long pulse widths generate collateraldestructive mechanical effects through intensive photodisruptiveprocesses. In addition, the application of ultraviolet radiation iscontroversial due to cytotoxic and mutagenic effects. UV radiation isalso absorbed outside the focus area by a plurality of endogenicmolecules. Accordingly, the success rate of this type of opticaltransfection is low. Testing of laser-assisted gene transfer has beenconducted since 1984 (Tsukakoshi et al. (1984) Appl. Phys. B35 135-140).Normally, ultraviolet (UV) nanosecond lasers with a relatively highpulse energy in the μJ range and single-shot mode are used. Thetransfection rates are very low, as is confirmed in the literature byTsukakoshi et al. (Appl. Phys. B35 (1984) 135-140) and Kurato et al.(Exp. Cell Res. 162 (1986) 372-378) which shows transfectionefficiencies of a maximum 0.6%. Tsukakoshi et al. describe anarrangement for the transfer of genes which is based on afrequency-tripled 10 Hz Nd:YAG nanosecond laser at a working wavelengthof 355 nm and which is outfitted, in addition, with a He—Ne laser aspilot laser and enables beam deflection through the use of twogalvoscanners. An image of the sample is displayed on a TV monitor bymeans of a transillumination apparatus using an UV blocking filter andis recorded by means of video recorders. Single shots with high pulseenergies of 1 mJ were used for perforating the cell membrane.

Nitrogen lasers with an emission wavelength of 337 nm were also used forgene transfer in plant cells (Weber et al.: Naturwissenschaften 75(1988) 36). In UV laser-assisted gene transfer in plant embryos, atransformation efficiency of 0.5% was reported (Greulich:“Micromanipulation by light in biology and medicine”, Birkhäuser Verlag,1999).

Another publication by Tao et al. (PNAS 84 (1987) 4180-4184) describesUV exposure with μJ pulses with a pulse duration of 10 ns in arelatively large irradiation area of 2.0-μm diameter using an invertedZeiss microscope with a 32× objective. At this magnification, thenumerical aperture is typically less than 0.8. The relatively largeirradiation spot involved probably caused a considerable perforation inthe membrane on the same order of magnitude. The transformationefficiency in these tests using human cells was less than 0.3%.

In sum, it can be stated that there are no known methods or apparatususable in practice for transferring molecules, particularly for theproduction of synthetic vaccines, into a specially selected individualcell efficiently and effectively without damaging the living cell.

OBJECT AND SUMMARY OF THE INVENTION

It is the primary object of the invention to find a novel possibilityfor targeted molecule transfer into the interior of vital cells,particularly the transfer of DNA, RNA, peptides, amino acids andproteins, which achieves a high transfer efficiency while extensivelyexcluding destructive side effects, such as a lethal effect, on atreated cell.

According to the invention, this object is met in a method fortransferring molecules into vital cells by means of laser radiation,preferably for targeted transfer of DNA, RNA, peptides, amino acids andproteins into vital cells and for the transfection of plant cells,animal cells and human cells, particularly for the production of drugssuch as synthetic vaccines, in that the cellular membranes are openedtransiently for the molecule transfer by multiple laser pulses in themicrojoule range or less, wherein a pulsed, near-infrared laser beamwith a pulse width in the femtosecond range is directed to asubmicrometer spot of a membrane of the vital cell for an irradiationperiod of less than one second.

Multiple laser pulses with a pulse repetition frequency in the MHz rangeor higher are advantageously used for laser irradiation. Laser pulseswith energies in the nanojoule range are preferably used. The laserpulses are advisably adjusted in such a way that a mean light intensityin the TW/cm² range is achieved in the laser spot on the membrane of thetarget cell.

It has proven advantageous for accuracy and for spatially limiting thetransient membrane permeability for molecular transfer to focus thelaser beam on a submicrometer spot in a diffraction-limited manner.

Further, for purposes of the technical implementation of the methodaccording to the invention in an arrangement for targeted transientmolecule transfer into vital cells by means of a laser beam,particularly for the transfer of DNA, RNA, peptides, amino acids orproteins, in which the molecules are moved from an extracellularenvironment through an optically increased permeability of a membraneinto the interior of an individual cell, with a laser whose laser beamis focused on the cell with a short pulse duration, the above-statedobject is met in that the laser is a mode-synchronized solid state laserwith an emission in the wavelength range of 700 nm to 1200 nm, thislaser being constructed as a pulse laser for generating multiplefemtosecond pulses with a pulse duration of less than 500 fs and pulseenergies in the microjoule range or less, in that focusing optics areprovided for preparing a diffraction-limited submicrometer spot, whereina light intensity in the terawatt per square centimeter range on thebiological membrane of a target cell is adjustable in the spot, in thata shutter for generating processing times of microseconds to a second isarranged in front of the laser, and in that devices for aligning andobserving the target cells are provided, wherein an additionalillumination source, imaging optics, special filters and a CCD camerafor finding and aligning a target cell and for detecting laser-induced,highly localized transient membrane changes is directed to the spotexposed by the laser, and a sample stage that is movable in threedimensions is provided for submicrometer-exact positioning of the targetcell relative to the laser spot and for focusing by means of az-adjustment unit.

The illumination source, in combination with the imaging optics and CCDcamera for target finding, target positioning and observation of thetransient membrane change, advantageously has a beam for transmittedlight illumination, preferably of white light.

In another advantageous variant, the illumination source has a beam forfluorescence excitation. Endogenic fluorophores (autofluorescence) orpreferably exogenic (administered) fluorophores (e.g., fluorescingmembrane markers) can be excited in this way.

Further, it has proven advisable when the laser beam is used at the sametime for two-photon excited fluorescence of a fluorescing membranemarker.

The laser beam advantageously has a pulse energy of less than 100 nm inthe laser spot on the target cell.

For sample handling with respect to the orientation of the laser spot onan individual target cell and a selected membrane surface, the laserbeam is focused on a fixed point; the positioning of the laser beamrelative to the target membrane is provided by means of the sample stageand the focus control is provided by means of the z-adjustment unit ofthe objective.

The shutter is advantageously switchable between a first opening statefor providing nanojoule pulses for membrane processing and a secondopening state for providing picojoule pulses for observing the beamposition.

In order to detect the position of the laser beam with respect to thetarget cell, the CD camera is advantageously outfitted with a filter sothat small portions of the laser beam reflected or transmitted by thetarget cell or its carrier are imaged on it and, further, has computerelements and control elements for showing the laser spot portionsimultaneously with the imaging of the target cell generated by the beamof the additional illumination source.

The image processing unit downstream of the CCD camera advisably has acomputer with special image processing software for identifying targetcells.

The computer is advantageously connected at the same time to controlunits and adjusting units for controlling the sample stage. Theinformation about target cells that is obtained from the objectidentification is provided for selecting and positioning the target celland for focusing the laser.

The control units and adjustment units are advisably provided on thebasis of the object identification supplied by the computer forpositioning a selected membrane of the target cell in the laser focus.

Further, the image processing unit advantageously contains imageprocessing software by which the course of the transient membranechanges can be detected. Accordingly, the successful molecule transfercan be detected and the search and processing of the next target cellcan be initiated automatically.

The basic idea of the invention consists in opening a membrane of avital target cell (prokaryot, eukaryot) transiently for the moleculetransfer through laser exposure with multiple laser pulses in themillijoule or nanojoule energy range in a submicrometer spot over anirradiation period of less than one second. In this way, in addition tothe external cell membrane, other intracellular membranes, e.g., thenuclear envelope, can be treated in a targeted manner.

It has been shown that by applying special laser irradiation of thiskind a laser-induced, highly localized transient membrane change throughcancellation of the barrier function in the area of the laser spot makespossible a temporary efficient transfer of molecules in the cell and,therefore, an efficient transfection without this molecule transferbeing accompanied by destructive side effects. The destructive sideeffects mentioned in the beginning, such as phototoxic effects and lowtransfection efficiency, do not occur.

The invention will be described more fully in the following withreference to an embodiment example.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings:

FIG. 1 is a schematic view of the arrangement according to theinvention; and

FIG. 2 illustrates the successful transfection of CHO cells withpEGFP-N1 in the form of an image acquisition of the expression a fewhours after irradiation by the intensive femtosecond pulses with CCDrecordings of single-photon and two-photon fluorescence imaging and NIRtransmission imaging.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention is described with respect to its process flow—withoutlimiting generality—with reference to an arrangement shown schematicallyin FIG. 1.

The arrangement basically comprises a laser 1 with a shutter 2 and beamexpander 3 arranged in front of it, an objective 5, a motor-operatedsample stage 6 with target cells 7 located thereon, which motor-operatedsample stage 7 supports the target cells so as to be movable relative tothe laser spot generated by the objective 5 by means of a z-adjustmentunit 8 of the objective 5 and an x,y,z-adjustment unit 9, an additionalillumination source 10, and a CCD camera 13 for recording an image ofthe treated target cells 7 that is generated by the illumination source10.

A mode-synchronized 80-MHz titanium-sapphire laser, as laser 1, iscoupled into a laser scanning microscope and is focused in adiffraction-limited manner on a submicrometer spot by means of a 40×objective 5 having the high numerical aperture of 1.3. The mean outputat a pulse width of about 200 fs is initially a few microwatts forobserving and searching the target cell. First, a cell layer of CHOcells (Chinese hamster cells), a cell layer of PTK cells (rat kangaroocells) and a cell layer of adult human DPSC stem cells were applied bymeans of a scanner and an image was generated based on the transmittedbeam and detection by means of a photomultiplier. In individual cases,the two-photon excited fluorescence signal of a fluorescing membranemarker can also be used for image generation. The cells are usuallylocated in a miniaturized sterile cell chamber containing 0.5 ml ofculture medium and 0.2 μg of plasmid DNA vector pEGFP-N1 (4.7 kb). Thisplasmid causes the synthesis of green fluorescing protein.

The laser beam is subsequently focused on a selected submicrometer areaof the membrane of an individual selected cell (scanner inpoint-irradiation mode) and the output is increased to between 50 mW and100 mW. The area is irradiated by means of a fast beam shutter for 16ms. Subsequent scans at a reduced average microwatt output showed theexistence of a transient membrane change (membrane eversion) in the areaof the selected membrane area of the target cell struck by the laserspot. An irradiation of this kind probably causes the temporarymicroperforation of the cell membrane through which the plasmid canpenetrate into the cell.

The irradiation mode was tested repeatedly on 200 cells. The timerequired for transfection through cell searching, beam focusing andirradiation was typically 10 to 15 seconds.

The integration of the DNA plasmid and the expression of the greenfluorescing protein was investigated in situ by stationary,time-resolved two-photon fluorescence imaging at an average laser outputof less than 1 mW over a duration of 72 hours. The successfullaser-induced expression of EGFP was confirmed by measuring the meanfluorescence lifetime of 2.4 ns. Regardless of cell type, it waspossible to achieve a transfection rate and an expression rate of morethan 90% as is shown impressively in FIG. 2.

The arrangement, according to the invention, for efficient moleculetransfer into an individual vital cell preferably contains amode-synchronized femtosecond laser with a high repetition frequencywhich is determined in the wavelength range between 700 nm and 1200 nmand, by means of an objective 5 with a high numerical aperture (greaterthan 0.8), provides a laser beam of multiple nanojoule laser pulseswhich is focused on a submicrometer spot in a rigidly fixed anddiffraction-limited manner with a light intensity in the TW/cm² rangefor transient cancellation of the barrier function of biologicalmembranes in the area of the laser spot. Further, the arrangementcomprises a fast shutter 2 which realizes irradiation periods in themicrosecond and millisecond range using nanojoule pulses and picojoulepulses for target adjustment, an additional illumination source 10,imaging optics 11, special filter 12 and CCD camera 13 for finding andadjusting the target and for detecting laser-induced, highly localizedtransient membrane changes, a preferably motor-operated sample stage 6with submicrometer accuracy, a z-adjustment unit 8, an image processingunit 15 with object identification and control modules for automatedtransfer of DNA, RNA and proteins into vital individual cells.

A femtosecond laser scanning microscope such as is known forapplications in the field of cell biology (see, e.g., Denk et al.Science 248 (1990) 73) is preferably used. In contrast to a beamdeflection (with special scanning optics, beam guidance and controls)that is usually carried out by means of a (costly) galvoscanner, thebeam of the femtosecond laser is localized on a fixed point by means ofbeam expander 3, deflecting unit 4 and objective 5 for scanning thesamples according to the invention. The scanning and focusing regimen isrealized solely by means of the motor-operated sample stage 6 and theadjustment units 8 and 9.

In a preferred construction in FIG. 1, a pulsed solid state laser withhigh beam quality (TEM₀₀ mode), an emission wavelength of 800 nm, highrepetition frequency (with typical values of around 80 MHz), a pulseduration of less than 300 fs, and a pulse energy of a few nanojoules(<100 nJ) is used as laser 1 for efficient molecule transfer. With afast shutter 2 with minimum switching times in the microsecond range,the laser beam can be released virtually without losses for treating thetarget cell 7 or can be blocked between 95% and 99% for the detection ofthe laser beam position in the target cell 7 before striking theexpander 3. The beam is then deflected to the focusing optics 5 with anumerical aperture of greater than 0.8, typically 1.2, by the deflectingmirror 4 (with a NIR reflectivity between 90% and 99%) and accordinglyfocused on a diffraction-limited submicrometer spot inside the sample 7located on the sample stage 6.

The sample, e.g., a vital cell—or, more simply, as target object: targetcell 7—is advisably located in a miniature cell chamber with at leastone glass window with a thickness of about 170 μm. The target cell 7 istypically surrounded by a medium which also contains molecules to betransferred, e.g., determined DNA plasmids. The laser beam is focusedthrough the glass window on a membrane section of the target cell 7.This membrane section can be the cell membrane, a cell wall, themembrane of an organelle or the nuclear envelope.

The focusing plane can be changed in depth (z-direction) with anaccuracy of less than 100 mm by means of a piezo-driven z-adjustmentunit 8. All three directions x, y, z of the positioning table 6 can beadjusted by the associated adjusting unit 9 with submicrometer accuracy(e.g., with integrated joystick).

The illumination source 10 of low light intensity ensures an imaging ofthe target cell 7 also during laser irradiation through the combinationwith imaging optics 11, short-pass filter 12, which causes a sharpattenuation of the laser radiation, and CCD camera 13. The actualposition of the laser spot, in addition to the visualization oftransmitted light of the illumination source 10, is received in such away that the laser beam reflected at the target cell 7 and/or its glasscarrier can be displayed with high spatial precision in the centralportion of the monitor 14 as a bright, non-halated laser spot togetherwith the image of the target cell 7 generated by the illumination source10. A computer, preferably a PC, as image processing unit 15 withcontrol unit and regulating unit and an image analysis program forobject identification (pattern recognition system) makes it possible todetect the target membrane and the automated displacement of the samplestage 6 and z-adjustment unit 8 in such a way that a part of the targetmembrane and the laser focus coincide. In fully-automatic orsemi-automatic operation, the shutter 2 is controlled immediately aftertarget adjustment and a large number of nanojoule laser pulses areapplied to a membrane of the target cell 7 for irradiation periods ofless than one second. Simultaneously and shortly after successful laserirradiation, a transient change in the membrane in the form of aneversion may be observed on the monitor 14. Normally, these membranechanges cease after several seconds to several minutes. Immediatelyafter irradiation, the membrane is permeable to molecules and enablesthe transfer of DNA, RNA and proteins in a particularly effectivemanner. When the transient change is detected by the image analysisprogram, a new target, usually a new target cell 7, is automaticallyadjusted by actuating the adjustment unit 9 by means of the controlmodule and the process described above is repeated. When no transientchange in the membrane is detected in the area of the irradiated field,the adjustment is carried out on a neighboring membrane region of thesame cell and laser irradiation is carried out again.

In the simplest case, the irradiation source 10 works with white light.However, it can also emit fluorescence excitation radiation instead ofwhite light, which makes it possible to detect fluorescing membranemarkers by means of CCD camera 13. The laser 1 itself can be used as afluorescence excitation source by means of two-photon effects. Inaddition, the described arrangement can also be integrated in amicroscope.

By target membrane is meant not only cell membranes or cell walls, butalso intracellular membranes such as nuclear envelopes and mitochondrialmembranes.

FIG. 2 shows the successful transfection of CHO cells with pEGFP-N1 andthe image recording of the expression several hours after irradiationwith the intensive femtosecond pulses through NIR transmissionrecording, two-photon fluorescence imaging and CCD display. The partialimages (scale: bar=25 μm) show:

-   a: a real EGFP fluorescence image by means of a CCD camera after    single-photon blue excitation; a number of fluorescing cells can be    seen after successful laser transfection;-   b: a NIR transmission image with an arrow indicating the individual    cells worked by the laser; and-   c: a two-photon fluorescence lifetime image in which the individual    fluorescing cell can be seen clearly after the laser processing;    surrounding cells which were not irradiated with the intensive laser    pulses do not fluoresce significantly; the calculated fluorescence    lifetime of 2.4 ns corresponds to the expected value for the green    fluorescing protein (GFP).

While the foregoing description and drawings represent the present itwill be obvious to those skilled in the art that various changes may bemade therein without departing from the true spirit and scope of thepresent invention.

Reference Numbers:

-   1 laser-   2 shutter-   3 expander-   4 deflecting mirror-   5 objective-   6 sample stage-   7 target cell (sample)-   8 z-adjustment unit-   9 adjustment unit-   10 (additional) illumination source-   11 imaging optics-   12 short-pass filter-   13 CCD camera-   14 monitor-   15 image processing unit

1-17. (canceled)
 18. A method for transferring molecules into vitalcells by laser radiation, preferably for targeted transfer of DNA, RNA,peptides, amino acids and proteins into vital cells and for thetransfection of plant cells, animal cells and human cells, particularlyfor the production of drugs such as synthetic vaccines, comprising thesteps of: opening cellular membranes transiently for the moleculetransfer by multiple laser pulses in the microjoule range or less, bydirecting a pulsed, near-infrared laser beam with a pulse width in thefemtosecond range to a submicrometer spot of a membrane of the vitalcell for an irradiation period of less than one second.
 19. The methodaccording to claim 18, wherein multiple laser pulses with a pulserepetition frequency in the MHz range or higher are used.
 20. The methodaccording to claim 18, wherein multiple laser pulses with energies inthe nanojoule range are used.
 21. The method according to claim 18,wherein laser pulses with a light intensity in the TW-cm² range areused.
 22. The method according to claim 18, wherein the laser beam isfocused on the submicrometer spot in a diffraction-limited manner. 23.An arrangement for targeted transient molecule transfer into vital cellsby means of a laser beam, particularly for the transfer of DNA, RNA,peptides, amino acids or proteins, in which the molecules are moved froman extracellular environment through an increased permeability of amembrane into the interior of a cell, with a laser whose laser beam isfocused on the cell with a short pulse duration, comprising: a laserwhich is a mode-synchronized solid state laser with an emission in thewavelength range of 700 nm to 1200 nm; said laser being constructed as apulse laser for generating multiple femtosecond pulses with pulsedurations of less than 500 fs and pulse energies in the microjoule rangeor less; focusing optics being provided for preparing adiffraction-limited submicrometer spot, wherein a light intensity in therange of terawatt per square centimeter on the biological membrane of atarget cell is adjustable in the spot; a shutter for generatingprocessing times of microseconds to a second being arranged in front ofthe laser; and devices for aligning and observing the target cells beingprovided, wherein an additional illumination source, imaging optics,special filters and a CCD camera for finding and aligning a target celland for detecting laser-induced, highly localized transient membranechanges is directed to the spot exposed by the laser; and a movablesample stage being provided for submicrometer-exact positioning of thetarget cell relative to the laser spot and for focusing by az-adjustment unit.
 24. The arrangement according to claim 23, whereinthe illumination source, in combination with the imaging optics and CCDcamera for target searching, target positioning control and observationof the transient membrane change, has a beam for transmitted lightillumination, preferably of white light.
 25. The arrangement accordingto claim 23, wherein the illumination source has a beam for fluorescenceexcitation, wherein endogenic fluorophores (autofluorescence) orpreferably exogenic (administered) fluorophores (e.g., fluorescingmembrane markers) can be excited in this way.
 26. The arrangementaccording to claim 23, wherein the laser beam is used at the same timefor two-photon excited fluorescence of exogenic fluorophores, preferablyfluorescing membrane markers.
 27. The arrangement according to claim 23,wherein the laser beam is focused on a fixed point, wherein thepositioning of the spot relative to the target membrane is provided bymeans of the sample stage and the focusing control is provided by meansof the z-adjustment unit of the sample stage.
 28. The arrangementaccording to claim 23, wherein the laser beam has a pulse energy of lessthan 100 nm in the laser spot on the target cell.
 29. The arrangementaccording to claim 28, wherein the shutter is switchable between a firstopening state for providing nanojoule pulses for membrane processing anda second opening state for providing picojoule pulses for observing thebeam position.
 30. The arrangement according to claim 23, wherein the CDcamera is outfitted with a filter so that small portions of the laserbeam reflected or transmitted by the target cell or by a carrier of thetarget cell are imaged on it and has an image processing unit forshowing the laser spot simultaneously with the imaging of the targetcell generated by the beam of the additional illumination source. 31.The arrangement according to claim 23, wherein the image processing unitdownstream of the CCD camera has a computer with special imageprocessing software for identifying target cells.
 32. The arrangementaccording to claim 31, wherein the computer is connected to controlunits and adjusting units for controlling the sample stage, wherein theinformation from the image processing and the object identification isprovided for selecting and positioning the target cell and for focusingthe laser.
 33. The arrangement according to claim 32, wherein thecontrol units and adjustment units are provided on the basis of theobject identification supplied by the computer for positioning aselected membrane of the target cell in the laser focus.
 34. Thearrangement according to claim 31, wherein the image processing unitcontains image processing software by which the course of the transientmembrane changes can be detected.